Ribonuclease H (endoribonuclease H, EC 5.1.26.4, hereafter referred to as RNase H) is an enzyme capable of hydrolyzing an RNA molecule when the RNA molecule is hybridized with a complementary DNA strand. The biological role of the enzyme is not known, and hence it is not known whether all organisms possess this enzyme.
RNase H is a useful tool in molecular biology research. RNase H is used for degrading the RNA strand after first-strand synthesis in the production of double-stranded cDNA. Okayama, H., et al. (1982) Mol. Cell. Biol. 2: 161-170.6; Gubler, U., et al. (1983) Gene 25: 263-269. The enzyme can remove poly-(A) tails from messenger RNA if the mRNA is reacted with oligo-dT.sub.12-18. Vournakis, J., et al. (1975) Proc. Natl. Acad. Sci. USA 72: 2959-2963; Davis, R., et al. (1988) Mol. Cell. Biol. 8: 4745-4755.
Most importantly, RNase H is used as a diagnostic tool for detecting specific target DNA sequences in a biological sample by a probe amplification process. Duck, P., et al. (1990) BioTechniques 9: 142-147. In this diagnostic technique, a probe is made of ribonucleoside bases flanked by deoxy-ribonucleoside bases. The probe hybridizes to a target DNA molecule. RNase H digests the ribonucleoside bases and cleaves the probe. The probe fragments then dissociate from the target. After many cycles, these fragments accumulate and serve as a basis for detecting the presence of the target.
RNase H is a component of another diagnostic test called the self-sustained sequence replication (3SR) amplification system, which is a transcription-based amplification method. Guatelli, J. C., et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878. In the 3SR system, a target nucleic acid sequence is replicated exponentially by using RNase H, a DNA-dependent RNA polymerase and reverse transcriptase.
RNase H may also be used to map the location of sequences on an RNA molecule. First the RNA is annealed with specific oligodeoxyribonucleotide probes and then the duplexed RNA is cleaved with RNase H. Donis-Keller, H. (1979) Nucleic Acids Res. 7: 179-192.
RNase H may also be used to quantitate poly-(A)-containing mRNA in biological samples. Krug, M. S., et al. (1987) Methods Enzymol. 152: 262-266. RNase H is useful in cDNA cloning via subtractive hybridization (Kzze, K., Shimizu, et al. (1989) Nucleic Acids Res. 17: 807) and for hybrid-arrest translation (Minshull, J., et al (1986) Nucleic Acids Res. 14: 6433-6451).
The RNase H enzyme used in the above-mentioned research was isolated from E. coli. The E. coli enzyme is called "RNase HI" and is the product of the rnhA gene. Berkower, I., et al. (1973) J. Biol. Chem. 248: 5914-5921; Kanaya, S., et al. (1983) J. Biol. Chem. 258: 1276-1281. RNase H has been identified in other organisms besides E. coli, such as yeast, KB cells, Krebs II ascites cells and avian myeloblastosis virus infected cells, although its existence in other species is still unknown. Crouch, R. J. (1981) in Gene Amplification and Analysis (Chirikjian, J. G., and Papas, T. S., eds.) Vol. 2, pp. 218-228, Elseivier, North Holland, N.Y.; Crouch, R. J., et al. (1982) in Nuclease (Linn, S. M., and Roberts, R. J., eds.) pp. 211-241, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. No RNase H has been isolated from any thermophilic organisms.
A thermostable RNase H would be preferable in many RNase H applications, particularly for mapping and certain diagnostic applications. In addition to the obvious advantages of longer reagent shelf life and greater stability under reaction conditions, a thermostable RNase H would allow reactions to be carried out at higher temperatures. These higher temperatures are closer to optimal temperatures for hybridization of RNA probes to target DNA and would destroy the activity of E. coli RNase H. The optimum temperature for a nucleic acid hybridization will depend on the hybridization buffer, but typically reaction temperatures in the 70.degree. C. to 95.degree. C. range facilitate maximum sensitivity by ensuring that target DNA sequences are melted and accessible and reaction temperatures in the 45.degree. C. to 75.degree. C. range facilitate maximum selectivity of hybrid formation because there is more hybridization stringency at higher temperature. High stringency conditions result in lower background by minimizing nonspecific binding of probes to unrelated target sequences.
What is needed in the art of molecular biology is an isolated RNase H capable of biological activity at elevated temperatures. Such an RNase H should be capable of biological activity after incubation at temperatures greater than 45.degree. C. for at least ten minutes. Preferably the RNase H will be capable of biological activity after incubation at temperatures of at least 70.degree. C. for at least ten minutes.